Homologous recombination is a pathway that cells use to repair double-stranded DMAbreaks (DSBs). Failure to repair DSBs results in gross chromosomal rearrangements, a hallmark of tumorogenesis, and several diseases, including many types of cancer, can result if this essential repair pathway is not working properly. The research goals presented here focus on understanding some early steps in the homologous recombination pathway. Particular emphasis will be placed on characterizing the reaction catalyzed by the human protein Rad51 and examining how this protein aligns homologous DMAmolecules. To facilitate this analysis we are developing unique systems for studying the real-time dynamics of DMA recombination at the level of single biochemical reactions using using total internal reflection fluorescence microscopy (TIRFM). The hypothesis behind the proposed research is that Rad51 aligns DMAsequences using a mechanism called intersegmental transfer. The specific aims are to: 1) Determine the mechanism by which the Rad51 and RecA proteins align DNA sequences. 2) Determine the temporal relationship between sequence alignment and other steps in the recombination reaction. 3) Determine how the DNA alignment process is affected in the context of chromatin.